The temporomandibular joint (TMJ) ankylosis is clinically defined as limited mouth opening due to either a fibrous or bony union between the head of the condyle and the glenoid fossa. Although most incidents of TMJ ankylosis occur after trauma or an infection, congenital cases have been reported. Currently little is known about the TMJ morphogenesis and the underlying genetic, cellular and molecular mechanisms. Particularly, nothing is known about genetic alteractions that cause congenital TMJ ankylosis. The homeobox gene Shox2 is expressed specifically in the mesenchymal cells of the maxilla-mandibular junction and later in the immatured chondrocytes of the condyle of the TMJ. A conditional inactivation of Shox2 in cranial neural crest derived cells leads to dysplasia and ankylosis of the TMJ. Thus, Shox2 mutant mice serve as a unique model system for the studies of the mammalian TMJ development and its underlying mechanisms. We hypothesize that Shox2 plays a crucial role in TMJ development by regulating Runx2 expression directly. We also hypothesize that SUMO modification of Shox2a is essential for the TMJ formation. Four specific aims are proposed to test these hypotheses. In Aim 1, we will determine if Runx2 is a direct downstream target of Shox2 by reporter assay, EMSA and ChIP assay. We will also determine if phosphorylation impairs Shox2a's transactivating potency by creating mutant forms of Shox2a mimicking constitutively phosphorylation state or constitutively dephosphorylation state. In Aim 2, we will define SUMO modification of Shox2a and its consequences on the modulation of the Shox2a's transcriptional capacity. In this aim, we will further define the interaction between Shox2a and Histone 3.3, which may enhance the transcription capacity of Shox2a. The human SHOXa will be included in parallel in the proposed studies. In Aim 3, we will determine the role of SUMO modifcation of Shox2a in the TMJ development by expressing the mutated forms of Shox2a that either can not be sumoylated or mimic constitutively sumoylated status in the endogenous Shox2-expressing domains. In the last aim, we will test if human SHOX and SHOX2 are functionally redundant in embryonic development through targeted insertion of the human SHOX gene into the mouse Shox2 allele. PUBLIC HEALTH RELEVANCE: The temporomandibular joint (TMJ) ankylosis is one of the major symptoms of TMJ disorders that occur highly frequently humans. TMJ ankylosis is clinically characterized by the formation of bone or fibrous adhesion of the joint components, which restricts the jaw movement and causes difficulty in feeding, swallowing, and breathing. Congenital TMJ ankylosis have been repored, but very little is known about the underlying genetic alteractions. A conditional inactivation of the homeobox gene Shox2 in the TMJ cells in mice leads to abnormal formation and ankylosis of the TMJ. Thus the conditional Shox2 mutant mice provide a unique animal model for studying TMJ development and TMJ ankylosis. This proposal studies the role of Shox2 in the TMJ development and its functional mechanisms using in vitro and in vivo approaches. The results obtained from this study will provide fundamental information for our understanding of the formation and function of this unique joint in humans.